Apparatus for purifying nucleic acid and method of purifying nucleic acid

ABSTRACT

A nucleic acid refining apparatus, being ease in automation thereof, keeping high in contacting frequency between nucleic acid within a sample and a solid phase during nucleic acid capture processing, thereby proving high capturing rate, comprises: means for separating a liquid containing the nucleic acid therein from said sample through centrifugal force; means for transferring a reagent through the centrifugal force; means for producing a mixture liquid of said reagent transferred through the centrifugal force and a solution containing said nucleic acid therein; a carrier for capturing said nucleic acid; means for transferring said mixture liquid to said carrier through the centrifugal force; heating means for heating said carrier; and a holding means for separating and holding the reagent containing said nucleic acid eluting from said carrier, separating from other reagent, through different centrifugal.

TECHNICAL FIELD

[0001] The present invention relates to an apparatus and a method forpurifying or refining nucleic acids therewith.

BACKGROUND ART

[0002] With advance of the molecular biology, various technologies aredeveloped in relation with genes, and with those technologies, it ispossible to separate and identify diseased genes, in much more numbersthereof. As a result, in the field of medical treatment, the molecularbiological technologies are introduced into diagnoses or checkingmethods, thereby enabling the diagnosis, which was impossible in theconventional art, and also achieving great reduction in the number ofdays necessary for the checks.

[0003] Such the advance is mainly owned to an amplification method of anucleic acid sequence, in particular, practicing of PCR (PolymeraseChain Reaction) technology or method. With this PCR method, it ispossible to amplify the nucleic acid in a solution, specifically upon asequence thereof. For this reason, it is possible to prove the existenceof a virus living in blood serum of an extremely small quantity oramount, indirectly, by amplifying the nucleic acid sequences as thegenes of the virus through the PCR method, for detection thereof.However, in a case where this PCR method is applied in daily clinicaldiagnoses in a clinical field, there still remain several problems to bedissolved. Among of them, a pre-treatment is very important, inparticular, for estimation by using the PCR method, i.e., the capacityof refining of the nucleic acid sequence, and for this reason, there areproposed several technologies or methods in relation to the refining ofthe nucleic acid sequence.

[0004] For a first technology relating to the conventional art, a methodis already disclosed, as described in Japanese Patent Laying-Open No.Hei 2-289596 (1990), for example, wherein silica particles, which cancombine with the nucleic acid sequence under existence of chaotropicmaterials, are used as a solid-phase for use of combining or couplingwith nucleic acid, for separating the solid-phase coupling with thenucleic acid sequence from a liquid, and thereafter complex of thesolid-phase and the nucleic acid sequence (solid-phase/nucleic acid) isrinsed, thereby eluting the nucleic acid sequence from the complex,depending upon necessity thereof.

[0005] Also, for a second technology relating to the conventional art, amethod is already disclosed, for example, in Japanese Patent Laying-OpenNo. Hei 8-320274 (1996), wherein a single nucleic acid sequence isextracted, collected, and/or separated, with using a large number ofcontainers for a single sample, a large number of detachable tips forseparate injection, filters, and particles of magnetic substance ormaterial.

[0006] Further, for a third technology relating to the conventional art,a method is already disclosed, for example, in Japanese PatentLaying-Open No. 2000-514928 (2000) of PCT application, wherein bloodplasma is separated from blood (i.e., whole blood) and further is mixedwith an extremely small quantity of liquid, as well as detection ofantibiotics, by using a microscopic channel, a chamber, a capillary, adeposable valve, etc.

[0007] However, the following problems can be listed, in the case wherethe methods or technologies disclosed in the conventional arts mentionedabove are applied, actually.

[0008] First of all, with the method described in the Japanese PatentLaying-Open No. Hei 2-289596 (1990), mentioned as the first technologyrelating to the conventional art in the above, since the nucleic acidsequence can not be collected within the same device, it is difficult inautomation thereof. Further, it is also difficult to increase thefrequency of contact between the nucleic acid and the silica particlesin a short time. In particular, in the case where the nucleic acid islow in the concentration, such as 10² copy/ml, for example, as a targetcontained within a sample, it is very difficult to increase the contactfrequency in a short time.

[0009] Also, with the method described in the Japanese PatentLaying-Open No. Hei 8-320274 (1996), mentioned as the second technologyrelating to the conventional art in the above, processes are complex forextracting, collecting, and/or separating of the nucleic acid sequences,therefore the automation is difficult. Further, with this conventionalart, since those processes cannot be treated within the same device, theproblem of contamination thereof may be worried about.

[0010] On the other hand, in the second conventional art, it isdisclosed that a pipette, to which the tip is connected, is linked witha cylinder (i.e., a syringe pump), so as to control thesuction/discharge amount by means of a servomotor or a pulse-motor,severely. For this reason, for maintaining sufficient suction/dischargeof the sample, it is impossible to increase the density of thesilica-membrane filter. On the other hand, if applying a low densitysilica-membrane filter having a superior penetration of liquid, as amatter of course, the probability of collecting the nucleic acid becomessmall. In particular, if the nucleic acid is low in the concentration,such as around 10² copy/ml, for example, as mentioned above, being thetarget contained within the sample, the contact frequency between thesilica-membrane filter and the nucleic acids is reduced further, whenthe sample passes through that silica-membrane filter.

[0011] Also, in the Japanese Patent Laying-Open No. 2000-514928 (2000)of PCT application, as the third conventional art, there is disclosedthe method for separating blood plasma from a very small amount ofblood, such as about 150 μL, for example. However, in this publication,nothing is disclosed therein, about a technology nor a method forrefining the nucleic acid.

[0012] Therefore, an object according to the present invention is toprovide an automatic refining apparatus and a method thereof, which canbe easily automated, wherein the contact frequency between the nucleicacids as the target within a sample and the solid-phase, therebyincreasing trapping rate or ratio of the nucleic acids, even under thecondition of the low concentration, such as around 10² copy/ml, forexample, and further to provide a structure for refining nucleic acidsfor building up thereof, and to provide a gene analyzing apparatusand/or a refining apparatus for chemical materials with use thereof.

DISCLOSURE OF THE INVENTION

[0013] According to the present invention, for achieving the objectmentioned above, there is provided a nucleic acid refining apparatus forrefining nucleic acid from a sample containing the nucleic acid therein,comprising: means for separating a liquid containing the nucleic acidtherein from said sample through centrifugal force; means fortransferring a reagent through the centrifugal force; means forproducing a mixture liquid of said reagent transferred through thecentrifugal force and a solution containing said nucleic acid therein; acarrier for capturing said nucleic acid; means for transferring saidmixture liquid to said carrier through the centrifugal force; heatingmeans for heating said carrier; and a holding means for separating andholding the reagent containing said nucleic acid eluting from saidcarrier, separating from other reagent, through different centrifugal.

[0014] Also, according to the present invention, there is provided anucleic acid refining apparatus for refining nucleic acid from a samplecontaining the nucleic acid therein, comprising: means for separating aliquid containing the nucleic acid therein from said sample throughcentrifugal force; reagent holding means for holding a reagent therein;means for transferring said reagent from said reagent holding meansthrough the centrifugal force; means for producing a mixture liquid ofsaid reagent transferred through the centrifugal force and a solutioncontaining said nucleic acid therein; a carrier for capturing saidnucleic acid; means for transferring said mixture liquid to said carrierthrough the centrifugal force; heating means for heating said carrier;and a holding means for separating and holding the reagent containingsaid nucleic acid eluting from said carrier, separating from otherreagent, through different centrifugal.

[0015] Also, according to the present invention, there is provided anucleic acid refining apparatus for refining nucleic acid from a samplecontaining the nucleic acid therein, comprising: a device having: meansfor separating a liquid containing the nucleic acid therein from saidsample through centrifugal force; means for transferring a reagentthrough the centrifugal force; means for producing a mixture liquid ofsaid reagent transferred through the centrifugal force and a solutioncontaining said nucleic acid therein; a carrier for capturing saidnucleic acid; means for transferring said mixture liquid to said carrierthrough the centrifugal force; heating means for heating said carrier;and a holding means for separating and holding the reagent containingsaid nucleic acid eluting from said carrier, separating from otherreagent, through different centrifugal; and a supply means for supplyingsaid reagent from an outside of said device.

[0016] Also, according to the present invention, there is provided anucleic acid refining apparatus for refining nucleic acid from a samplecontaining the nucleic acid therein, comprising: a round front surfacecover having a hole, which is sealed by a rubber at one end thereof; agap defined between said front surface cover; a first round disc andhaving a separation gel, which lies in said gap, for separating asolution including nucleic acid therein from said sample and a groove,which lies in said gap, for quantification of said solution; a secondround disc having: a reagent reservoir containing a reagent therein; aflow passage; a carrier for combining the nucleic acid thereon; anelution reservoir for accumulating an eluting solution, being obtainedfrom said reagent after eluting the nucleic acid therein; and a wasteliquid reservoir, being provided following said eluting solutionreservoir, for accumulating the reagent other than said elutingsolution, and having a flow passage being opened into an outside; and around reverse surface cover having a heating body, wherein a device isbuilt up with said reverse surface cover, said second disc, said firstdisc and said front surface cover, being piled up sequentially, and hasa punching portion, through which said solution and said reagent canmove in a direction of thickness of said device, by punching a hole at apredetermined position within said device.

[0017] Further, according to the present invention, in the structure ofthe nucleic acid refining apparatus as described in the above, wherein a“U” shaped flow passage is provided between said elution solutionreservoir and said waste liquid reservoir on said second disc, orwherein a branch passage, being divided in the direction of thickness ofsaid device, is provided between said elution solution reservoir andsaid waste liquid reservoir on said second disc, or wherein a filter,having lower penetrability than that of said carrier, is providedbetween said elution solution reservoir and said waste liquid reservoiron said second disc.

[0018] And, according to the present invention, also for achieving theobject mentioned above, there is provided a method for refining nucleicacid from a sample containing the nucleic acid therein, comprising thefollowing steps of: a step for separating a solution containing nucleicacid therein from said sample through centrifugal force, with using aseparation gel, within a first gap provided within an inside of adevice, being formed with laminating a plural number of round discs; astep for quantification of said solution with provision of a first holewithin said device; a step for transferring said quantified solutioninto a flow passage, as a second gap formed within said device, withprovision of a second hole within said second device; a step fortransferring a combining liquid of a first reagent into said flowpassage through centrifugal force, with provision of a third hole withinsaid device; a step for producing a mixture liquid of said solutionquantified within said flow passage and said combining liquid; a stepfor passing said combining liquid through said carrier for capturingsaid nucleic acid thereon through centrifugal force, therebytransferring it into a waste liquid reservoir as a third gap formedwithin said device; a step for transferring a rinsing liquid of a secondreagent into said flow passage through the centrifugal force, withprovision of a fourth hole within said device; a step for making saidrinsing liquid pass through said carrier through the centrifugal force,thereby transferring it into said waste liquid reservoir; a step fortransferring an eluting solution of a third reagent into said flowpassage through the centrifugal force, with provision of a fifth holeformed within said device; a step for letting said carrier hold saideluting solution therein, and heating said carrier; a step fortransferring said eluting solution containing said nucleic acidseparated from said carrier therein into an eluting solution reservoirformed within a flow passage following said carrier, through thecentrifugal force, thereby holding the eluting solution thereinseparating from other reagents; and a step for collecting said elutingsolution from said eluting solution reservoir, from an outside of saiddevice.

[0019] Also, according to the present invention, there is provided anucleic acid refining structure, being formed to be rotatable,comprising: a supply portion, through which is supplied a liquidincluding nucleic acid therein; a nucleic acid capture portion, ontowhich is captured the nucleic acid within said supplied liquid; awashing liquid supply portion, through which a washing liquid issupplied to said nucleic acid capture portion; a waste portion, intowhich is wasted said washing liquid flowing through said nucleic acidcapture portion; an eluting solution supply portion, through which aneluting solution is supplied into said nucleic acid capture portion; andan eluting solution holder portion for holding the eluting solutionincluding the nucleic acid therein, being captured on said nucleic acidcapture portion once and separated therefrom, after flowing through saidnucleic acid capture portion, wherein said eluting solution hold portionis formed within a flow passage communicating between said nucleic acidcapture portion and said waste portion.

[0020] Also, according to the present invention, there is provided anucleic acid refining structure, being formed to be rotatable,comprising: a supply portion, through which is supplied a liquidincluding nucleic acid therein; a nucleic acid capture portion, ontowhich is captured the nucleic acid within said supplied liquid; awashing liquid supply portion, through which a washing liquid issupplied to said nucleic acid capture portion; a waste portion, intowhich is wasted said washing liquid flowing through said nucleic acidcapture portion; an eluting solution supply portion, through which aneluting solution is supplied into said nucleic acid capture portion; andan eluting solution holder portion for holding the eluting solutionincluding the nucleic acid therein, being captured on said nucleic acidcapture portion once and separated therefrom, after flowing through saidnucleic acid capture portion, wherein said eluting solution holderportion is formed in a downstream of said nucleic acid capture portion,while said waste portion in a downstream of said eluting solution holderportion.

[0021] Also, according to the present invention, there is provided anucleic acid refining structure, being formed to be rotatable,comprising: a supply portion, through which is supplied a liquidincluding nucleic acid therein; a nucleic acid capture portion, ontowhich is captured the nucleic acid within said supplied liquid; awashing liquid supply portion, through which a washing liquid issupplied to said nucleic acid capture portion; a waste portion, intowhich is wasted said washing liquid flowing through said nucleic acidcapture portion; an eluting solution supply portion, through which aneluting solution is supplied into said nucleic acid capture portion; andan eluting solution holder portion for holding the eluting solutionincluding the nucleic acid therein, being captured on said nucleic acidcapture portion once and separated therefrom, after flowing through saidnucleic acid capture portion, wherein said eluting solution holderportion is formed on an outer periphery side of said nucleic acidcapture portion, while said waste portion on an outer periphery side ofsaid nucleic acid holder portion.

[0022] Also, according to the present invention, there is provided anucleic acid refining structure, being formed to be rotatable,comprising: a supply portion, through which is supplied a liquidincluding nucleic acid therein; a nucleic acid capture portion, ontowhich is captured the nucleic acid within said supplied liquid; awashing liquid supply portion, through which a washing liquid issupplied to said nucleic acid capture portion; a waste portion, intowhich is wasted said washing liquid flowing through said nucleic acidcapture portion; an eluting solution supply portion, through which aneluting solution is supplied into said nucleic acid capture portion; aneluting solution holder portion for holding the eluting solutionincluding the nucleic acid therein, being captured on said nucleic acidcapture portion once and separated therefrom, after flowing through saidnucleic acid capture portion; and a waste liquid flow passagecommunicating between said eluting solution holder portion and saidwaste portion, wherein said waste liquid flow passage comprises a holderportion communication portion being communicated with an area, beinglocated on an outer periphery side rather than an area on a most-innerperiphery side of said eluting solution holder portion, an innerperiphery side area portion, being located in a downstream of saidcommunication portion and in an inner periphery side rather than saidcommunication portion, and a waste portion communication portion, beinglocated in a downstream of said inner periphery side area portion andbeing communicated with said waste portion, being located in an outerperiphery side rather than said inner periphery side area portion.

[0023] Also, according to the present invention, there is provided anucleic acid refining structure, being formed to be rotatable,comprising: a supply portion, through which is supplied a liquidincluding nucleic acid therein; a nucleic acid capture portion, ontowhich is captured the nucleic acid within said supplied liquid; awashing liquid supply portion, through which a washing liquid issupplied to said nucleic acid capture portion; a waste portion, intowhich is wasted said washing liquid flowing through said nucleic acidcapture portion; an eluting solution supply portion, through which aneluting solution is supplied into said nucleic acid capture portion; aneluting solution holder portion for holding the eluting solutionincluding the nucleic acid therein, being captured on said nucleic acidcapture portion once and separated therefrom, after flowing through saidnucleic acid capture portion; and a waste liquid flow passagecommunicating between said eluting solution holder portion and saidwaste portion, wherein a connection portion between said elutingsolution holder portion and said waste liquid flow passage is formed inan outer periphery side rather than a most-inner periphery portion ofsaid waste liquid flow passage, and in an inner periphery side ratherthan a most-outer periphery portion of said waste liquid flow passage.

[0024] Also, according to the present invention, there is provided anucleic acid refining structure, being formed to be rotatable,comprising: a supply portion, through which is supplied a liquidincluding nucleic acid therein; a nucleic acid capture portion, ontowhich is captured the nucleic acid within said supplied liquid; a firstreagent supply portion, through which a first reagent is supplied tosaid nucleic acid capture portion; a waste portion, into which is wastedsaid first reagent flowing through said nucleic acid capture portion; asecond reagent supply portion, through which a second reagent issupplied to said nucleic acid capture portion, having a function ofreleasing said nucleic acid captured on said nucleic acid captureportion therefrom, being larger than that of said first reagent; asecond reagent holder portion for separating the nucleic acid capturedon said nucleic acid capture portion therefrom, thereby to hold saidsecond reagent contained within an inside thereof; and a waste liquidflow passage communicating between said second reagent holder portionand said waste portion, wherein an area located on said waste flowpassage between a most-inner periphery portion and a connection portionwith said second reagent holder portion, and an area located on an outerperiphery side than said most-inner periphery portion of said secondreagent holder portion are formed, so that a total volume of them issmaller than that of said first reagent to be supplied, while beinglarger than that of said second reagent to be supplied.

[0025] Also, according to the present invention, there is provided anucleic acid refining structure, being formed to be rotatable,comprising: a supply portion, through which is supplied a liquidincluding nucleic acid therein; a nucleic acid capture portion, ontowhich is captured the nucleic acid within said supplied liquid; awashing liquid supply portion, through which a washing liquid issupplied to said nucleic acid capture portion; a waste portion, intowhich is wasted said washing liquid flowing through said nucleic acidcapture portion; an eluting solution supply portion, through which aneluting solution is supplied into said nucleic acid capture portion; aneluting solution holder portion for holding the eluting solutionincluding the nucleic acid therein, being captured on said nucleic acidcapture portion once and separated therefrom, after flowing through saidnucleic acid capture portion; and a waste liquid flow passagecommunicating between said eluting solution holder portion and saidwaste portion, wherein an area located on said waste flow passagebetween a most-inner periphery portion and a connection portion withsaid eluting solution holder portion and an area located on an outerperiphery side than said most-inner periphery portion of said elutingsolution holder portion are formed, so that a total volume of them issmaller than that of said washing liquid to be supplied, while beinglarger than that of said eluting solution to be supplied.

[0026] Also, according to the present invention, there is provided anucleic acid refining apparatus comprising: a receiver portion forreceiving said nucleic acid refining structure described in the above;and a rotary driving mechanism for rotating said nucleic acid refiningstructure.

[0027] Also, according to the present invention, there is provided thenucleic acid refining apparatus as described in the above, furthercomprising an analysis mechanism for analyzing genes of said nucleicacid, with using said nucleic acid introduced into said eluting solutionholder portion of said nucleic acid refining structure.

[0028] Also, according to the present invention, there is provided thenucleic acid refining apparatus as described in the above, furthercomprising a heating device for heating said nucleic acid introducedinto said eluting solution holder portion of said nucleic acid refiningstructure.

[0029] Also, according to the present invention, in the nucleic acidrefining apparatus as described in the above, wherein said washingliquid is controlled, so as to be held within said waste portion, beingsupplied from said nucleic acid capture portion through said elutingsolution holder portion, in an amount larger than that a total volume ofan area located on said waste flow passage between a most-innerperiphery portion and a connection portion with said eluting solutionholder portion and an area located on an outer periphery side than saidmost-inner periphery portion of said eluting solution holder portion,while said eluting solution is controlled, so as to be held within saideluting solution holder portion, being supplied through said nucleicacid capture portion, in an amount smaller than that the total volume ofthe area up to the connection portion between the most-inner peripheryportion of said waste flow passage and said eluting solution holderportion and the area located on the outer periphery side than saidmost-inner periphery portion of said eluting solution holder portion.

[0030] And, according to the present invention, there is furtherprovided a chemical material refining structure comprising: a supplyportion, through which is supplied a liquid including a first chemicalmaterial therein; a first chemical material capture portion, onto whichis captured said chemical material within said supplied liquid; a firstreagent supply portion, through which a first reagent is supplied tosaid first chemical material capture portion; a waste portion, intowhich is wasted said first reagent flowing through said first chemicalmaterial capture portion; a second reagent supply portion, through whicha second reagent is supplied to said first chemical material captureportion, having a function of releasing said chemical material from saidfirst chemical material capture portion, being larger than that of saidfirst reagent; and a second reagent holder portion for holding saidsecond reagent including said first chemical material therein, beingcaptured on said first chemical material capture portion once and thenseparated therefrom after flowing through said first chemical materialcapture portion, wherein said second reagent holder portion is formedwithin a flow passage communicating between said first chemical martialholder portion and said waste portion.

[0031] Furthermore, in the present invention, though the rinsing liquidis described to be a fluid having a function of removing impuritiesother than the nucleic acid included within said nucleic acid holderportion, while the eluting solution to be a liquid having a function ofseparating the nucleic acid from said capture portion, therebycontaining the nucleic acid in itself, however it is not used to belimited only to that having such the function of eluting, and it has alarge function of separating the nucleic acid from said capture portion,rather than that of the rising liquid.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1 is a perspective view of a disc device, being an embodimentaccording to the present invention;

[0033]FIG. 2 is a plane view of a cover on a front surface-side thereof,in the disc device according to the present invention;

[0034]FIG. 3 is a plane view of a first (1^(st)) layer disc on a frontsurface-side thereof, in the disc device according to the presentinvention;

[0035]FIG. 4 is a plane view of a second (2^(nd)) layer disc on a frontsurface-side thereof, in the disc device according to the presentinvention;

[0036]FIG. 5 is a plane view of the second (2^(nd)) layer disc on areverse surface side thereof, in the disc device according to thepresent invention;

[0037]FIG. 6 is a plane view of the cover on a reverse surface sidethereof, in the disc device according to the present invention;

[0038]FIG. 7 is an a-a′ cross-section view of the disc device shown inFIG. 1 mentioned above;

[0039]FIG. 8 is a plane view of the first-layer disc mentioned above,showing a step for separating blood serum from blood with using a gel,in the disc device according to the present invention;

[0040]FIG. 9 is the a-a′ cross-section view of the disc device shown inFIG. 1 mentioned above, showing the condition of separation of the bloodserum from blood;

[0041]FIG. 10 is the a-a′ cross-section view of the disc device shown inFIG. 1 mentioned above, showing a step for removing the blood serum inexcess;

[0042]FIG. 11 is the a-a′ cross-section view of the disc device shown inFIG. 1 mentioned above, showing a step for taking out the blood serumwhich is quantified;

[0043]FIG. 12 is the a-a′ cross-section view of the disc device shown inFIG. 1 mentioned above, showing a step for passing or penetrating amixture, mixing up the quantified blood serum and a combining liquidtherein, through a carrier;

[0044]FIG. 13 is a plane view of the front side surface of the seconddisc, showing a step for transferring the liquid mixture and rinsingliquid into a water liquid reservoir;

[0045]FIG. 14 is the a-a′ cross-section view of the disc device shown inFIG. 1 mentioned above, showing a step for eluting nucleic acid into aneluting solution through transferring the liquid to the position of theeluting solution;

[0046]FIG. 15 is the a-a′ cross-section view of the disc device shown inFIG. 1 mentioned above, showing a step for collecting the elutingsolution having passed through the carrier;

[0047]FIG. 16 is a flowchart for showing a method for refining nucleicacids according to the present invention;

[0048]FIG. 17 is a cross-section view of a disc device, for explainingabout the structure of the disc device and the method for refining thenucleic acids, of other (second; 2^(nd)) embodiment according to thepresent invention, in particular, corresponding to the a′-a′cross-section view shown in FIG. 1 mentioned above;

[0049]FIG. 18 is the a-a′ cross-section view of the disc device, showinga step for collecting the eluting solution in the disc device accordingto the second embodiment;

[0050]FIG. 19 is a plane view for showing the front surface-side of thesecond-layer disc of the disc device, explaining about the structure ofthe disc device and the method for refining the nucleic acids, offurther other (third; 3^(rd)) embodiment according to the presentinvention;

[0051]FIG. 20 is a view for showing an outlook of the nucleic acidrefining apparatus, into which is applied the disc device according tothe present invention mentioned above;

[0052]FIG. 21 is a total structure view of a gene analyzing apparatus,according to an embodiment of the present invention;

[0053]FIG. 22 is a perspective and explosive view of an analyzer disc ofthe gene analyzing apparatus mentioned above;

[0054]FIG. 23 is a perspective view for showing flow path within theanalyzer disc mentioned above;

[0055]FIG. 24 is a flowchart for showing flows of extracting andanalyzing operations with the analyzer disc mentioned above;

[0056]FIG. 25 is also a flowchart for showing flows of extracting andanalyzing operations with the analyzer disc mentioned above;

[0057]FIG. 26 is a view for showing the flowing condition of the liquid,which contains the sample (i.e., the blood) therein, within a flowpassage of the analyzer disc mentioned above;

[0058]FIG. 27 is also a view for showing the flowing condition of theliquid, which contains the sample therein, within a flow passage of theanalyzer disc mentioned above;

[0059]FIG. 28 is further also a view for showing the flowing conditionof the liquid, which contains the sample therein, within a flow passageof the analyzer disc mentioned above;

[0060]FIG. 29 is further also a view for showing the flowing conditionof the liquid, which contains the sample therein, within a flow passageof the analyzer disc mentioned above;

[0061]FIG. 30 is further also a view for showing the flowing conditionof the liquid, which contains the sample therein, within a flow passageof the analyzer disc mentioned above;

[0062]FIG. 31 is further also a view for showing the flowing conditionof the liquid, which contains the sample therein, within a flow passageof the analyzer disc mentioned above;

[0063]FIG. 32 is further also a view for showing the flowing conditionof the liquid, which contains the sample therein, within a flow passageof the analyzer disc mentioned above;

[0064]FIG. 33 is further also a view for showing the flowing conditionof the liquid, which contains the sample therein, within a flow passageof the analyzer disc mentioned above;

[0065]FIG. 34 is further also a view for showing the flowing conditionof the liquid, which contains the sample therein, within a flow passageof the analyzer disc mentioned above;

[0066]FIG. 35 is further also a view for showing the flowing conditionof the liquid, which contains the sample therein, within a flow passageof the analyzer disc mentioned above;

[0067]FIG. 36 is further also a view for showing the flowing conditionof the liquid, which contains the sample therein, within a flow passageof the analyzer disc mentioned above;

[0068]FIG. 37 is further also a view for showing the flowing conditionof the liquid, which contains the sample therein, within a flow passageof the analyzer disc mentioned above;

[0069]FIG. 38 is further also a view for showing the flowing conditionof the liquid, which contains the sample therein, within a flow passageof the analyzer disc mentioned above;

[0070]FIG. 39 a view for showing an example of the structure fordetecting the rotation position of a holder disc, according to theembodiment mentioned above; and

[0071]FIG. 40 is a view for showing operational waveforms of examples ofoperation timings of a drilling machine.

BEST MODE FOR CARRYING OUT THE INVENTION

[0072] Hereinafter, explanation will be given on embodiments accordingto the present invention, by referring to drawings attached herewith.

[0073] Herein, first of all, as one embodiment according to the presentinvention will be explained a disc-type device, which is applied forrefining a so-called ribonucleic acid (i.e., RNA), such as, the HCVvirus, the HIV virus, etc., included in blood (i.e., whole blood), forexample. Explanation will be given on the structure of a deviceaccording to the first embodiment, by referring to FIGS. 1 to 7, and inaddition thereto, explanation will be given on a method for refining theRNA with use thereof, by referring to FIGS. 8 to 15 and 16. Also, byreferring to FIGS. 17 and 18, explanation will be given on the structureof other embodiment (i.e., a second structure) according to the presentinvention, and a method for refining the RNA with use thereof.Furthermore, by referring to FIG. 19, explanation will be given on thestructure of further other embodiment according to the present invention(i.e., a third structure) according to the present invention, and amethod for refining the RNA with use thereof, and by referring to FIG.20 will be explained an apparatus applying the disc device of thepresent invention therein; thus, the refining apparatus of nucleicacids.

[0074]FIG. 1 is a perspective view for showing an outlook of the discdevice, constituting a central portion of the refining apparatus ofnucleic acids, according to the present invention. The disc device 1 isbuilt up with covers on a front surface side and a reverse surface side,both being in disc-like in the configuration thereof, and further alaminated structure of two (2) layers disposed therebtween. In moredetails, it is build up with a front surface-side cover 2, a first-layerdisc 3, a second-layer disc 4, and a reverse surface-side cover 5. Also,in the front surface-side cover 2 are formed a large number of ports,including a blood inlet port 6 and an eluting solution collection port13 for collecting the eluting liquid, within a liquid of which thenucleic acids are purified or refined, etc. Further, the disc device 1is fixed onto a support shaft 50 for use of rotation of the disc device.Further, the support shaft 50 for use of rotation of the disc device isconnected to, such as an electric motor, etc., thereby driving the discdevice 1 rotationally.

[0075]FIG. 2 shows the plane structure of the front surface-side cover2, being a constituent part of the disc device 1 mentioned above,according to the present invention. On this front surface-side cover 2,as shown in the figure, are formed four (4) quadrants (4 sectors), beingdisposed symmetrically in positions, each including a blood inlet port6, a valve (a) opening port 7, a valve (b) opening port 8, a valve (c)opening port 9, a valve (d) opening port 10, a valve (e) opening port11, a valve (f) opening port 12, and a port 13 for use of collection ofeluting solution, respectively. Further, as is shown in FIG. 2 (also, inthe same manner as in FIG. 1), in each of the four (4) quadrants areformed ports a-f, respectively, but detailed explanation will be givenonly on the ports of the first quadrant (sector A), as a representativeone thereof. However, other sectors, i.e., the (sector B), the (sectorC) and the (sector D), are also same to that of the sector A, thereforethe detailed explanation thereof will be omitted herein.

[0076] In FIG. 2, each of the ports, being attached with referencenumerals 6-13, is built up with a film-like sheet made of a thickrubber, such as a silicon rubber, etc., for example, being stuck orfirmly fixed with an adhesive or the like, thereby forming a coverthrough deposing it into bores formed on the front surface-side cover 2.However, a needle is stuck through the rubber onto the film-like sheet,on which that rubber is fixed, and thereafter, in a case where theneedle is pull out, the opening portion formed by sticking of the needleis closed up with the self-restoring force due to elasticity, which isinherently owned by the rubber. For this reason, there is no chance thatthe liquid remaining within the disc device 1 during the rotation of thedisc device 1 comes out from the opening portion to be scattered into anoutside thereof.

[0077]FIG. 3 shows the plane structure of the first-layer disc 3, as aconstituent of the disc device 1 mentioned above, according to thepresent invention. This first-layer disc 3 is partitioned symmetrically,thereby to form four (4) chambers (sectors A to D), being similar in theshape thereof, as shown in the figure. In each of those chambers areprovided a hollowed separation air gap 16 formed, a gutter (a recessportion) 17, being formed in one body together with the separation airgap 16 but hollowed deeper than that, and plural partition walls 15,each having the same height to that of the outer periphery portion ofthe disc (herein, each chamber has five (5) pieces of walls, therebybeing divided into six (6) small chambers). Within each of the smallchambers in about “U” shape, being defined between the partition wall 15and the outer periphery portion mentioned above, a gel 14 is providedtherein, respectively (i.e., six (6) gels in each chamber), and also avalve (a) 18 for use of transferring the serum in excess and a valve (b)19, which are formed on the bottom of the gutter 17, for use oftransferring the serum accumulated within the gutter 17 are provided inthe center of the first-layer disc 3. In a middle portion thereof areformed a needle insertion bore 20 for opening the valve (c), a needleinsertion bore 21 for opening the valve (d), a needle insertion bore 22for opening the valve (e), and a needle insertion bore 23 for openingthe valve (f), i.e., provided as those separated from the separation airgap 16, and also a tip insertion bore 24 for collecting the solution ina part of the partition wall 15.

[0078] As was mentioned in the above, with the disc device 1 accordingto the embodiment of the present invention, since four (4) chambers (aneluting chamber) are provided separately, within an inside thereof, itis possible to introduce four (4) kinds of bloods at the same time.However, the number of those eluting chambers should not be restrictedonly thereto, but it may be less or grater than that. Also, the valves(a) 18 and (b) 19 are those, which are normally closed in the conditionthereof. Further, those valves are formed in one body together with thefirst-layer disc 3 and those portions are formed to be very thin in thethickness thereof, comparing to the other portions (however, explanationwill be given later on the cross-sectional construction of the disc).

[0079]FIG. 4 shows the structure on a front surface-side of thesecond-layer disc 4, being the constituent part of the disc, accordingto the present invention. In this second-layer disc 4, in the samemanner as in the first-layer disc 3 mentioned above, the chambers (thesectors) are partitioned symmetrically, thereby forming four (4)chambers being same in the shape thereof. And, in each of the chambers(the sectors) are provided an excess serum reservoir 25 for introducingthe excess serum in separating operation of blood cells, a combiningliquid reservoir 26 for accumulating a combining liquid (a reagent forcombining the nucleic acid contained within the blood serum with thesolid phase), which is provided in a central portion of the excess serumreservoir 25, reservoir 27 for a rinsing liquid A and reservoir 28 for arinsing liquid B for accumulating rinsing liquids (a rinsing liquid Aand a rinsing liquid B: reagents for rinsing the dirty solid phaseattaching components thereon, including protein components containedwithin the blood serum and the combining liquid, without taking out thenucleic acid combining on said the solid phase therefrom), and aneluting solution reservoir 29 for accumulating the eluding liquid (areagent for eluting the nucleic acid combining onto the solid phase),respectively.

[0080] And, on this second-layer disc 4 are also provided a serumpenetration opening 30, through which moves the blood serum obtained bymeans of the first-layer disc 3, in an inner peripheral directionthereof, and a valve (c) 31 for transferring the combining liquid,within the combining liquid reservoir 26 in the outer peripheraldirection thereof. Further, within the reservoir 27 for rinsing liquidA, a valve (d) 32 is provided for transferring the rinsing liquid Aremained within the reservoir 27 of rinsing liquid A, in the outerperipheral direction thereof, and also within the reservoir 28 forrinsing liquid B, a valve (e) 33 for transferring the rinsing liquid Bremained within the reservoir 28 for rinsing liquid B, in the outerperipheral direction thereof.

[0081] Also, within the eluting solution reservoir 29, there is provideda valve (f) 34 for transferring the eluting liquid, in the outerperiphery direction thereof, and in the outer periphery portion of thedisc is formed a tip insertion portion 35 for use of collecting theeluting liquid, being connected with the tip insertion bore 24 for useof collection of eluting liquid, which is formed in the first-layer disc3 mentioned above.

[0082] As mentioned above, with the disc device 1, according to thepresent invention, also four (4) chambers are formed in the second-layerdisc 4, locating below the first-layer disc 3, corresponding to those inthe first-layer disc 3. Also, the valve (c) 31, the valve (d) 32, thevalve (e) 33, and the valve (f) 34 are in the closed condition,normally. Further, those valves are formed together with thesecond-layer disc 4 in one body, and those portions are very thin in thethickness thereof, comparing to the other portions.

[0083]FIG. 5 shows the structure on the reverse side surface of thesecond-layer disc 4 mentioned above, as the constituent element of thedisc device 1 according to the present invention. On the reverse sidesurface of the second-layer disc 4, as shown in the figure, there areprovided a serum discharge outlet 40, through which a quantified bloodserum can flows out, and flow passages 36 for achieving a role oftransferring the rinsing liquid A, the rinsing liquid B and the elutingliquid, respectively. Also, on the reverse side surface of thesecond-layer disc 4 is provided a carrier 37 made of high-densitysilica, as the solid phase for combining with the nucleic acid, throughwhich are transferred the mixture between the blood serum and thecombining liquid, the rinsing liquid A and the rinsing liquid B, while avalve 41 for use of collecting the eluting liquid is provided in thereservoir of collected eluting liquid, which is formed in a partthereof, for maintaining the eluting liquid therein. Further, there arealso provided a flow passage 38 formed in “U” shape, through whichpasses the mixture of the blood serum and the combining liquid, therinsing liquid A and the rinsing liquid B, a waste liquid reservoir 39for accumulating the mixture of the blood serum and the combiningliquid, the rinsing liquid A and the rinsing liquid B therein, and aflow passage 45 for use of removing air, being provided for dischargingthe air remaining within the waste liquid reservoir 39.

[0084] Also, in each chamber (the sector) formed on the reverse sidesurface of the second-layer disc 4 are provided a trap, provided forprohibiting the components, such as, the serum, the combining liquid,the rinsing liquid A, the rinsing liquid B, etc., from scattering in theform of mist thereof into an outside of the disc 1, i.e., as a mistscattering prevention filter 43, formed in a part thereof, and an outerconnecting hole 44 being connected to an outside of the disc device 1,respectively.

[0085]FIG. 6 shows the structure on a front surface-side cover 5, as theconstituent part of the disc device 1, according to the presentinvention. On the front surface-side cover 5, as shown in the figure, isformed an outer connection bore 44 at the same position to that of thesecond-layer disc 4. Also at the position corresponding to the carrier37 provided on the second-layer disc 4 mentioned above, in particularbelow there, resistors 46 for use of heating the carrier are disposed,each of which generates heat due to conduction of current therethrough,thereby heating up the region or area of the carrier 37 up to apredetermined temperature. Further, there are provided wirings 47 forconnecting those resistors for use of heating the carrier, electricallyin series, and also electrode pads for connecting with outer terminalsof the disc device 1, being made up with so-called a plus (+) electrode48 and a minus (−) electrode 49, for conducting the electricity throughthe four (4) resistors 46 for heating the carrier.

[0086] Next, the cross-section views of the disc 1 mentioned above areshown, by referring to FIGS. 7, 9, 10, 11, 12, 14, and 15. FIG. 7 showsthe a-a′ cross-section view of the disc device 1 shown in FIG. 1mentioned above. Also, FIG. 7 corresponds to the a-a′ cross-sectionviews shown in FIGS. 2, 3, 4, 5, and 6 mentioned above, respectively.Namely, the disc device 1 is made up with lamination of the frontsurface-side cover 2, the first-layer disc 3, the second-layer disc 4,and the reverse surface-side cover 5, in that order thereof. In moredetails, the disc device 1 is built up by laminating those, i.e., thefront surface-side cover 2 shown in FIG. 2 mentioned above, thefirst-layer disc 3 shown in FIG. 3 mentioned above, the second-layerdisc 4 shown in FIG. 4 mentioned above, the reverse-side of thesecond-layer disc shown in FIG. 5 mentioned above, and the reversesurface-side cover 5 shown in FIG. 6 mentioned above, in that order,sequentially.

[0087] On the a-a′ cross-section of the front surface-side cover 2 inFIG. 7 are formed an opening port 7 of the valve (a), an opening port 8of the valve (b), an opening port 9 of the valve (c), an opening port 12of the valve (f), and a collection port 13 for eluting liquid,respectively. Also, on the first-layer disc 3, the cross-section ofwhich is shown in this a-a′ cross-section view, are provided aseparation gap 16 gutter 16 formed with a gutter 17, a valve (a) 18, anda valve (b) 19, and further are formed a needle insertion bore 20 foruse of opening the valve (c), being provided with connecting to theopening port 9 of the valve (c), a needle insertion bore 23 for use ofopening the valve (f), being provided with connecting to the openingport 12 of the valve (f), and a tip insertion bore 24 for use ofcollecting the eluting liquid, being provided with connecting to thecollecting port 13 of the eluting liquid, respectively.

[0088] Also on the second-layer disc 4, shown in a-a′ cross-sectionview, are provided and/or formed an excess serum reservoir 25, acombining liquid reservoir 26, an eluting solution reservoir 29, a serumpenetration opening 30, a valve (c) 31, a valve (f) 34, a tip insertionportion 35 for collecting the eluting liquid, being provided withconnecting to the tip insertion portion 24 for use of collecting theeluting liquid, a flow passage 36 for mixing, a carrier 37, a wasteliquid reservoir 39, a valve 41 for use of collecting the elutingliquid, and a collected eluting liquid reservoir 42, respectively.

[0089] On the reverse side-surface cover 5 shown in a-a′ cross-sectionview is formed the resistors 46 for heating the carrier. Further, thedisc device 1, being built up by fixing the above-mentioned frontsurface-side cover 2, the first-layer disc 3, the second-layer disc 4,and the reverse surface-side cover 5, is fixed, connecting onto asupporting shaft 50 for rotating the disc device. In more details, thisdisc device 1 is fixed, connecting onto the supporting shaft 50, so thatthe center thereof comes to be on the central axis of rotation of thesupporting shaft 50 for rotating the disc device.

[0090] The disc device 1 is built up with such structure as mentionedabove. However, also herein, other structures, such as the valve (a) 18,the valve (b) 19, the serum penetration opening 30, the valve (c) 31,the valve (f) 34, and the valve 41 for collecting the eluting liquid,for example, are explained only in the portion of the sector C, forescaping from duplicate of explanation thereof. However it is needlessto say that they are totally same with, at the position correspondingthereto the sector A.

[0091] Next, explanation will be given on a method for purifying orrefining nucleic acids from a sample, in more details, for refining theRNA of the HCV virus, HIV virus, etc., from blood, by using the discdevice 1, the structure of which was explained in the above.

[0092] First of all, explanation will be made on a step for separatingthe serum from the blood by using the disc device 1. FIG. 8 shows thestandstill condition of the first-layer disc 3, where it is stopped inrotation, after injecting the blood containing the target virus thereininto the inside of the first-layer disc 3 by sticking the needle tubeinto the blood insertion port 6 on the front surface-side cover 2, androtating the disc device 1 by the motor for about five (5) minutes so asto generate the centrifugal force of around 2,000 G.

[0093] Under this condition, in each chamber, so-called a blood cells51, including a red corpuscle, which is the highest density, and/or awhite corpuscle, etc., fills up the outermost periphery portion, withinthe separation gap 16 thereof. On the other hand, the blood serum 52including the virus therein fills up the gutter 17 provided within theseparation gap 16 at the central portion of the disc, thereby being in arelationship of disposition where the gel 14 lies between them. This isbecause the blood is separated by the centrifugal force appliedthereupon, due to the rotation of the disc device 1. In more details,because the density of the gel 14 is lower than that of the blood cells51 and is higher than that of the serum components including the virus,therefore the blood cells 51, where the centrifugal force is applied,shifts or moves to the outermost periphery portion thereof, forcedlypassing through an inside of the gel 14, which is provided on theoutermost periphery portion of the separation gap 16 mentioned above.

[0094] Further, since no such the power is applied onto the blood cells51 or the like, any more, corresponding to the centrifugal force appliedby the rotation, when the disc device 1 is stopped in the rotationthereof, therefore none of the blood cells 51 or the like passes throughwithin the gel 14, thereby moving to the central portion of the disc.For this reason, the blood cells 51 or the like will never be mixed upwith the serum 52 containing the virus therein, even when the discdevice 1 is stopped in the rotation thereof. However, it is preferableto determine the length of the said partition wall 15 in advance, sothat the gel 14 will stop at a predetermined position of the partitionwall 15 after completing the series of operations of rotation/stoppingwith respect to the disc device 1.

[0095]FIG. 9 shows the a-a′ cross-section of the disc device 1, underthe condition shown in FIG. 8 mentioned above. Within the separation gap16 of the firs-layer disc 3 is filled up the serum 52 containing thetarget virus therein, and within the gutter 17 is also filled up theserum 52, in the same manner. Also, within the combining liquidreservoir 26 is filled up the combining liquid, and further within theeluting solution reservoir 29 is filled up the eluting solution.Further, though not shown in this FIG. 9, within the rinsing liquid Areservoir 27 is filled up the rinsing liquid A, and within the rinsingliquid B reservoir 28 is filled up the rinsing liquid B. Herein, thecombining liquid, the eluting solution, the rinsing liquid A, and therinsing liquid B are held within the second-layer disc 4 in advance,however the present invention should not be restricted onto to such thestructure. For example, the combining liquid, the eluting solution, therinsing liquid A, and the rinsing liquid B may be injected into thecombining liquid reservoir 26, the eluting solution reservoir 29, therinsing liquid A reservoir 27, and the rinsing liquid B reservoir 28mentioned above, through an external mechanism, such as a pipetteattached with a needle tube, for example, through the valve (c) openingport 9, the valve (f) opening port 12, the valve (d) opening port 10(not shown in the figure), or the valve (e) opening port 11 (not shownin the figure), respectively and/or every time, depending upon thenecessity or corresponding to the processing thereof.

[0096] Next, FIG. 10 is a view for explaining the processing fortransmitting the serum other than the necessary amount thereof into theexcess serum reservoir 25 provided in the second-layer disc 4, withusing a needle 53. This processing is as follows:

[0097] (1) The needle 53 is inserted into the disc 1 stopped in therotation thereof, through the valve (a) opening port 7;

[0098] (2) The needle 53 is pulled out after breaking through the valve(a) 18 with that needle 53; and

[0099] (3) The serum in excess flows into the excess serum reservoir onthe second-layer disc 4.

[0100] At the same time, the serum is accumulated within the gutter 17provided in the separation gap 16, in the necessary amount thereof.

[0101] By the process as mentioned above, the quantified serum can beobtained in the necessary amount thereof, and on the other hand, theserum in excess is discharged to an outside of the first-layer disc 3.However, the valve (a) opening port 7, which was opened once throughsticking of the needle 53, will be closed up through the self-restoringforce due to the elasticity of that rubber, as mentioned above. Also,the volume of the gutter 17 is determined to become the volume, wherethe serum can enters therein in the necessary amount in advance.

[0102] Next, FIG. 11 is a view for explaining the processing fortransmitting the necessary amount of serum containing the virus thereinfrom the first-layer disc 3 to the mixture flow passage 36 provided inthe second-layer disc 4, with using the needle 53. This process is asfollows:

[0103] (1) The needle 53 is inserted into the disc 1 through the valve(b) opening port 8;

[0104] (2) The needle 53 is pulled out after breaking through the valve(b) 19 with that needle 53; and

[0105] (3) By means of the gutter 17 provided in the separation gap 16,the quantified serum 54 containing the virus therein passes throughserum penetration opening 30, and flows into the mixture flow passage 36on the second-layer disc 4, thereby accumulating therein.

[0106] By the process mentioned above, the quantified serum 54 istransferred into the mixture flow passage 36. However, also the valve(b) opening port 8, which was opened once through sticking of the needle53, will be closed up through the self-restoring force due to theelasticity of that rubber.

[0107] Next, FIG. 12 is a view for explaining the process for elutingthe virus into the mixture obtained by mixing the quantified serum 54containing the virus therein (but, not shown in this FIG. 12) and thecombining liquid (not shown in the figure), producing the mixture liquid55 into which the RNA of that virus is eluted, and further penetratingthat mixture liquid through the carrier 37 made of the high densitysilica through the centrifugal force, thereby catching or capturing theRNA of that virus eluted by combining it with the carrier 37. Further,this processing is as follows:

[0108] (1) The needle 53 is inserted into the disc device 1 through thevalve (c) opening port 9;

[0109] (2) The needle 53 passes trough the valve (c) opening needleinsertion bore 20, thereby breaking through the valve (c) 31. And, theneedle 53 is pulled out;

[0110] (3) The combining liquid held within the combining liquidreservoir 26 flows into the mixture flow passage 36. In this instance,the disc device 1 is rotated at a low rotation speed, to generate suchan amount of centrifugal force, that the mixture liquid of thequantified serum 54 containing the virus therein and the combiningliquid cannot pass through the carrier 37. With this, the combiningliquid within the combining liquid reservoir 26 passes through the valve(c) 31 due to this centrifugal force, and all of it flows down into themixture flow passage 36;

[0111] (4) For mixing up the quantified serum 54 with this combiningliquid, upon the disc device 1 is given a rotation in theforward/reverse direction, periodically, for a certain time period, suchas several seconds, for example. With such the operation of the discdevice 1 as was mentioned above, the combining liquid and the quantifiedserum 54 containing the virus therein are mixed up, thereby producingthe mixture liquid 55 into which RNA of the virus is eluted; and

[0112] (5) Upon this mixture liquid 55 is applied the centrifugal force,so that it can pass through the carrier 37 made of the high densitysilica, such as about 30,000 G, for example. Since this carrier 37 madeof silica is high in the density, it is high in the ratio of contact,and therefore the RNA can be easily caught or captured with the carrieronly if the mixture liquid 55 containing the RNA therein passestherethrough.

[0113] By the process as mentioned above, the mixture liquid 55containing the RNA therein passes through the carrier 37 made of highdensity silica, thereby capturing the RNA by means of the carrier 37.Also herein, the valve (c) opening port 9, which was opened once throughsticking of the needle 53, will be closed up through the self-restoringforce due to the elasticity of that rubber.

[0114] Next, FIG. 13 is a view for showing the process for transferringthe mixture liquid 55 indicted by hutching into the waste liquidreservoir 39 due to the principle of the centrifugal force and thesiphon's law. This processing is as follows:

[0115] (1) The disc device 1 is rotated at a rotation speed, so as togenerate the centrifugal force, such as about 10,000 G, for example, sothat the mixture liquid 55 penetrates through the carrier 37,completely, and that no mixture liquid remains within the carrier 37. Inthis instance, the mixture liquid 55 remains within a portion, which isbuilt up with the “U”-shaped flow passage 38 connected to the wasteliquid reservoir 39, the collected eluting liquid reservoir 42, and theeluting liquid collecting valve 41, and filled up with therein;

[0116] (2) Penetrating through the “U”-shaped flow passage 38 by thecentrifugal force of about 10,000 G mentioned above, the mixture liquid55 is transferred into the waste liquid reservoir 39, gradually. In thisinstance, due to the movement of the mixture liquid 55, the air iscompressed within the waste liquid reservoir 39, and this air isdischarged into an outside of the disc device 1, through the air removalflow passage 45, the mist scattering prevention filter 43, through whichthe air can pass, and the outer connection bore 44. In this manner, themovement of the mixture liquid 55 can be achieved, smoothly; and

[0117] (3) Since the mixture liquid 55 is filled up within the“U”-shaped flow passage 38 and the collected eluting liquid reservoir42, under the condition containing no air-layer therein, all the mixtureliquid 55 is transferred into the waste liquid reservoir 39 due to thesiphon's law, without remaining liquid therein. In the manner as wasmentioned in the above, the mixture liquid 55 is transferred into thewaste liquid reservoir 39. However, the mixture liquid 55 has such anamount thereof, that it fills up within the spaces of the “U”-shapedflow passage 38 and the collected eluting liquid reservoir 42 mentionedabove, under the condition of containing no air-layer therein, oralternatively the “U”-shaped flow passage 38 and the collected elutingliquid reservoir 42 are designed, so as to satisfy such the condition.

[0118] Next, though not shown in the figure, after transferring themixture liquid 55 into the waste liquid reservoir 39, a step or processis carried out of rinsing or washing away components of the protein andthe combining liquid, which adhere onto the carrier 37. This iscompleted, by breaking through the valve (d) 32 and the valve (e) 33with using the needle 53, and by applying the centrifugal force to therinsing liquids A and B, respectively, so as let the rinsing liquids Aand B penetrate through the carrier 37, in the same manner as in thetransfer of the combining liquid. In this instance, the proteincomponent is removed away by means of the rinsing liquid A, while thecombining liquid component by means of the rising liquid B. Further, allthe rinsing liquid A and the rising liquid B are transferred into thewaste liquid reservoir 39, due to the same principle or law to that,which is applied in the step or process for transferring the mixtureliquid 55 into the waste liquid reservoir 39.

[0119] Next, FIG. 14 is a view for showing the processing fortransferring the eluting solution 56 into the carrier 37 with using theneedle 53, thereby eluting the RNA of virus combining with the carrier37 into the eluting solution 56. This processing is as follows:

[0120] (1) The needle 53 is inserted into the disc device 1 through thevalve (f) opening port 12;

[0121] (2) Passing through the valve (f) opening needle insertion bore23, the needle 53 breaks through the valve (f) 34. And, thereafter, theneedle 53 is pulled out;

[0122] (3) The eluting solution 56 held within the eluting solutionreservoir 29 flows into the mixture flow passage 36. In this instance,the disc device 1 is rotated at a low rotation speed, so that theeluting solution 56 cannot penetrate through the carrier 37. With this,the eluting solution 59 contained within the eluting solution reservoir29 passes through the valve (f) 34 due to the centrifugal force, and allof it flows down into the mixture flow passage 36. And, with thiscentrifugal force, the eluting solution 56 comes to be in contact withthe carrier 37, as shown in the figure, and after the carrier 37 isimmersed therein, the disc device 1 is stopped in the rotation thereof;and

[0123] (4) Conducting electricity through the carrier heating resistors46, the carrier 37 is heated up together with the eluting solution 56held in an inside thereof, i.e., from 60° C. to 70° C. in temperature.By the process as mentioned above, the eluting solution 56 istransferred to the carrier 37, thereby eluting the RNA combining withthe carrier 37 into the eluting solution 56. Further, in this instance,the valve (f) opening port 12, which was opened once through sticking ofthe needle 53, will be closed up through the self-restoring force due tothe elasticity of that rubber.

[0124] Next, FIG. 15 is a view for showing the processing forholding/collecting the eluting solution 56, into which the RNA of virusis eluted, with using an eluting solution collection chip 57 attachedwith a needle tube. This processing is as follows:

[0125] (1) The eluting solution 56, into which is eluted the RNA held onthe carrier 37 in the previous step or process, is moved into thecollected eluting liquid reservoir 42 due to the centrifugal force, suchas of around 3,000 G, for example, and it is held at that position;

[0126] (2) The eluting solution collection chip 57 is inserted into thedisc device 1 through the eluting solution collection port 13;

[0127] (3) This eluting solution collection chip 57 passes through theeluting liquid collecting tip insertion portion 24 and the elutingliquid collecting tip insertion portion 35, and breaks through theeluting liquid collecting valve 41; and

[0128] (4) The eluting solution collection chip 57 is held at theposition of the collected eluting liquid reservoir 42, and the elutingsolution 56 into which the RNA is eluted is absorbed into this elutingsolution collection chip 57, thereby being collected therein.

[0129] By the process mentioned above, the collection of the elutingsolution 56 is achieved, into which the RNA of virus is eluted. Alsoherein, the eluting solution collection port 13, which was opened oncethrough sticking of the eluting solution collection chip 57, will beclosed up through the self-restoring force due to the elasticity of thatrubber. Also, the eluting solution 56 is determined in a liquid amountthereof, so that it is not enough for filling the “U”-shaped flowpassage 38 shown in FIG. 5 mentioned above, but sufficient for fillingthe collected eluting liquid reservoir 42 partially. In other words, the“U”-shaped flow passage 38 and the collected eluting liquid reservoir 42mentioned above are designed so that they are in such the condition asmentioned above. For this reason, the eluting solution 56 will not betransferred into the waste liquid reservoir 39 shown in FIG. 5, even ifthe centrifugal force, such as about 10,000 G mentioned above, isapplied to the eluting solution 56, into which the RNA is eluted.Passing through each of the steps or processes mentioned above, from theblood, it is possible to refine the RNA of the virus contained therein.

[0130] Herein, in FIG. 16 attached are shown the refining processes ofsteps mentioned above, being collected together, in the form of a flowthereof. Namely, a step 1 is that for conducting theseparation/quantification of serum. In this step, a blood is injectedinto from the blood insertion port 6, and the disc is rotated toseparated the blood through the centrifugal force by rotating thereof,thereby holding the quantified serum 54 of the necessary amount in thegutter 17 (corresponding figures: FIGS. 8, 9, and 10).

[0131] A step 2 is a step for combining the nucleic acid. In this step,the quantified serum 54 within the gutter 17 mentioned above moves intothe mixture flow passage 36, while the combining liquid for combiningwith the nucleic acid moves into the mixture flow passage 36, and thenthe quantified serum 54 and the combining liquid are mixed up therein.And, those mixture liquid 55 penetrates through the carrier 37, duringthe process of which the nucleic acid (i.e., the RNA) combine with thecarrier 37. And the mixture liquid 55 flows into the waste liquidreservoir 39 (see, corresponding figures: FIGS. 11, 12, and 13).

[0132] A step 3 is a rinsing process for the carrier. In this step, therinsing liquid A and the rinsing liquid B penetrate through the carrier37, and move into the waste liquid reservoir 39.

[0133] And, a step 4 is a separation/collection process of the nucleicacid. In this process, the eluting solution 56 moves within the mixtureflow passage 36, and this eluting solution 56 is held by means of thecarrier 37. Then, the eluting solution 56 is heated through the heatingof the carrier heating resistors 46. Thereafter, this eluting solution56 moves into the collected eluting liquid reservoir 42, and iscollected from the eluting solution collection port 13 (see,corresponding figures: FIGS. 14 and 15).

[0134] Next, explanation will be given on the structure of the discdevice 1, according to other embodiment (i.e., the second embodiment) ofthe present invention, and a refining method with using thereof,hereinafter.

[0135] The structure of the disc device 1 according to the secondembodiment differs from that of the first embodiment, only in thestructure of the second-layer disc 4 thereof. In more details, thoughnot shown in FIG. 17, it differs from that mentioned above, in thestructure and the position a waste liquid reservoir 59 for accumulatingwaste liquid including the quantified serum 54 and the combining liquid,and the rinsing liquid A and the rinsing liquid B. Further, it alsodiffers therefrom, in that a branch flow channel 58 branched is providedin the downstream flow path after the carrier 37 mentioned above.

[0136] Next, explanation will be made on the process for refining theRNA from blood, by referring to FIGS. 17 and 18. In the same manner asin the first embodiment (i.e., the first method) mentioned above, theserum in excess is discharged with using the needle 53 (however, notshown in those FIGS. 17 and 18), and the mixture liquid 55 including thequantified serum 54 containing the virus therein and the combiningliquid 55 (after mixing, into this mixture liquid 55 is eluted the RNAthereof, being dissolved from the virus), is brought to penetratethrough within the carrier 37 by the centrifugal force of about 3,000 G.However, in this instance, the mixture liquid 55 penetrating through thecarrier 37 is transferred up to the position of the collected elutingliquid reservoir 42, due to this centrifugal force, once.

[0137] Further, due to the centrifugal force of about 10,000 G, themixture liquid 55 comes up through the branch flow channel 58 and itreaches to the waste liquid reservoir 59 formed on the surface side ofthe second-layer disc 4. However, in this instance, since the branchflow channel 58 is formed in such the shape as shown in the figure, theliquid will not turns back to the collected eluting liquid reservoir 42(see FIG. 17), if the disc device 1 is stopped in the rotation thereof.Furthermore, regarding the rinsing liquid A and the rinsing liquid B,they are also made to penetrate through the carrier 37, and thereby aretransferred into the waste liquid reservoir 59, in the same principle.Next, in the same manner to the method of the first embodiment mentionedabove (i.e., the first method), the valve (f) 34 is opened by the needle53, thereby letting the carrier 37 to hold the eluting solution 56therein, and further thereafter, the eluting solution 56 and the carrier37 are heated by means of the carrier heating resistors 46. Then, theeluting solution 56 comes up from 60° C. to 70° C. in the temperaturethereof, and thereby allowing the carrier 37 to elute the RNA of viruscombining thereon into the eluting solution 56.

[0138] Thereafter, the eluting solution 56 penetrates through within thecarrier 37 due to the centrifugal force, and is transferred into thecollected eluting liquid reservoir 42. In this instance, to the elutingsolution 56 is applied the centrifugal force, so that the said elutingsolution 56 will not move up through the branch flow channel 58 to thewaste liquid reservoir 59, however the eluting solution 56 can penetratethrough the carrier 37 (such as, the centrifugal force of about 3,000 G,for example). Finally, in the same manner to that of the firstembodiment mentioned above (i.e., the first method), the elutingsolution collection chip 57 is inserted through the eluting solutioncollection port 13 and the eluting liquid collecting tip insertionportion 24, thereby collecting the eluting solution 56, into which theRNA is eluted. With such the structure and the steps as mentioned above,it is possible to refine the target RNA.

[0139] Next, explanation will be given on the structure of the discdevice, according to further other embodiment (i.e., the thirdembodiment) of the present invention, and a refining method with usingthereof.

[0140]FIG. 19 shows the structure on the surface side of thesecond-layer disc 4 building up the disc deice 1. The structures ofother parts than that are same with those in the first embodimentmentioned above, therefore the explanations thereof will be omittedherein.

[0141] Further, with the disc device 1 according to the further otherembodiment (i.e., the third embodiment), it differs from the firstembodiment mentioned above, in the structure that, within the flowpassage connecting from the carrier 37 to the waste liquid reservoir 39is provided a filter 61, which has the density being further higher thanthat of the carrier mentioned above, and that the collected elutingliquid reservoir r 42 is formed in a drum-like shape so that it caneasily hold the eluting solution 56 therein (not shown in FIG. 19). Withthe second-layer disc 4 having such the form as mentioned above, aftermaking the mixture liquid 55 containing the quantified serum 54including the virus therein and the combining liquid (after mixingthereof, into the mixture liquid is eluted the RNA of virus throughdissolving of the virus) penetrate through within the carrier 37, themixture liquid 55 is further brought to penetrate through the filter 61mentioned above due to the centrifugal force, such as of about 20,000 G,for example, thereby being transferred into the waste liquid reservoir39. Thereafter, bringing also the rinsing liquid A and the rinsingliquid B to penetrate through the carrier 37, sequentially, on the sameprinciple mentioned above, thereby they are transferred into the wasteliquid reservoir 39. Finally, applying the centrifugal force of such thedegree, that it can penetrate through the carrier 37 but cannot passthrough the filter 61 (such as, the centrifugal force of about 3,000 G,for example), the eluting solution 56, into which the RNA of virus iseluted, is accumulated within the collected eluting liquid reservoir 42.And, the eluting solution 56 is collected in the same manner as in thefirst method mentioned above.

[0142] Namely, with such the structures and the steps mentioned above,it is possible to refine the RNA in the disc device 1 according to thethird embodiment.

[0143] Finally, explanation will be made on the method for refining thenucleic acid as the target, i.e., the RNA of virus, from the blood asthe sample, with using a nucleic acid refining apparatus being made upwith the disc device 1, according to the present invention, thestructures and the operations of which was explained above in thedetails thereof.

[0144] First, FIG. 20 is a diagrammatical view for showing a briefconcept of the nucleic acid refining apparatus with using the discdevice 1. This apparatus comprises: a motor 62 connected to the discdevice 1; an arm 63 attached with an electricity conduction terminal forconducting electricity to the disc device 1; a punching machine 64 foropening the valve(s) within an inside of the disc device 1; a robot arm65 attached with a chip for collecting the eluting solution, into whichthe RNA is eluted; a pump 66 for sucking out the eluting solution;piping 67; an eluting solution collection bottle 68 to be injected withthe collected eluting solution; a robot arm 69 attached with a chip forblood for injecting the blood into the disc device 1; a blood bottle 70for holding the blood to be tested therein; and a housing 71. However,for the apparatus shown herein, it is not always necessary to bestructured in the above manner, nor to be structured with all theconstituent elements thereof, and in the place thereof, for the personskilled in the art, various kinds of variations can be considered inmany ways.

[0145] However, the steps of the method for refining the RNA are asfollows, with using the apparatus, an example of the structure of whichis shown in FIG. 20.

[0146] (1) The sample blood is taken out by a certain amount thereof,through the pump 66, from the blood bottle 70 for holding the bloodincluding the virus as the target therein, by means of the robot arm 69attached with the chip for blood, and it is injected into the inside ofthe disc device 1 through the blood insertion portion 6;

[0147] (2) Passing through the steps of the disc device 1 explainedabove, sequentially (i.e., blood-cell separation; mixture of the serumand the combining liquid; penetration of the mixture liquid through thecarrier; penetration of the rinsing liquids through the carrier; elutionof the RNA combining with the carrier; and holding the eluting solution,separating it from the mixture liquid and the rinsing liquids), theeluting solution 56 can be obtained, into which the RNA of virus iseluted.

[0148] However, herein, the valve (a) 18, the valve (b) 19, the valve(c) 31, the valve (d) 32, the valve (e) 33, and the valve (f) 34 areopened by means of the punching machine 64 mentioned above. Also, thisdisc device 1 is rotated by means of the motor 62. When conducting theelectricity through the carrier heating resistors 46, the disc device 1is stopped in the rotation thereof, and the arms 63 attached with theelectricity conduction terminal are contacted with a plus (+) electrode48 and a minus (−) electrode 49 of the disc device 1;

[0149] (3) The eluting liquid collecting valve 41 within the inside ofthe disc device 1 is opened by means of the robot arm 65 attached withthe chip for collecting the eluting solution, thereby collecting theeluting solution 56, into which the RNA is eluted, by means of the pump66. Further, that eluting solution 56 is moved into the eluting solutioncollection bottle 68, thereby obtaining the eluting solution 56 desired.

[0150] In the manner mentioned above, it is possible to refine only theeluting solution containing the RNA therein, from the blood includingthe RNA as the target, separating it from co-existing materials includedwithin the blood.

[0151] The blood (i.e., the whole blood) was explained as the samplecontaining the nucleic acid therein, in the explanations given in theabove. However as the sample containing such the nucleic acid therein,others may be included than the blood (i.e., the whole blood), forexample, vital samples, such as, the blood serum and urine, etc., andalso biological samples, such as, cultivated cells and/or bacteria,etc., and the present invention can be applied thereto. Also, as thetarget nucleic acid, the explanation was given on the HCV virus or theHIV virus as the one example thereof, however according to the presentinvention, it should not be restricted only to those, but it may beapplied to others, such as, deoxyribonucleic acid (DNA), for example.Further, with the silica-made carrier made for catching the nucleic acidmentioned above may be made up by using silica particle, quartz wool,quartz filter paper, or fragmentation thereof, for example. Also, as thecombining liquid containing the chaotropic ion therein, the solution ofguanidine thiocyanate is preferable, however other than this, it may bethe solution of guanidine-hydrochloric acid, the solution of sodiumiodide, or the solution of potassium iodide, etc. Further, as the risingliquid A is preferable a solution mainly containing guanidinethiocyanate therein, for example, or as the rising liquid B ispreferable a water solution containing 50% of ethanol therein. And, asthe eluting solution, TE buffer solution (pH 8.0) is preferable.

[0152] Also, according to the present invention, it is possible toincrease contact frequency between the nucleic acid within the sampleand the solid phase, even in the case when concentration of the nucleicacid contained within the sample is low, such as of 10² copy/ml about,and with this, it is possible to refine the nucleic acid at highcollection rate. Also, since the steps mentioned above are processedwithin the inside of the same device, according to the presentinvention, the problem of contamination can be dissolved.

[0153] Further, according to the embodiment of the present invention, itis possible to make the refining operation of the nucleic acid automate,easily. Furthermore, according to the present invention, since thecentrifugal force can be applied to the refining processes of thenucleic acid, it is possible to increase the density of the solid phaseprovided within the carrier, therefore the nucleic acid can be refinedat high collection rate, even in the case of low concentration of thenucleic acid contained within the sample.

[0154] Next, explanation will be given on other embodiment of thenucleic acid refining apparatus, according to the present invention, byreferring to FIGS. 21 to 38.

[0155]FIG. 21 shows the total structure of a gene analyzer with usingthe nucleic acid refining apparatus according to the present invention.This gene analyzer 901 comprises: a holder disc 912 being supportedrotatably by a motor 911; plural analysis discs 902 of a sector form,each being positioned on the holder disc 912 by means of projections121; a punching machine 913 for controlling the flow of liquids; and two(2) sets of optical apparatuses, i.e., an upper portion opticalapparatus 914 and a lower portion optical apparatus 915. Further, theholder disc 912 comprises a holder disc optical window 122 for the lowerportion optical apparatus 915.

[0156]FIG. 22 is a view for showing the refining structure of nucleicacid, i.e., the structure of an analysis disc 902. This analysis disc902 is basically constructed by connecting between an upper cover 920and a channel portion 930. The upper cover 920 comprises: a sampleinjection opening 210; a plural number of reagent injection openings220, 230, 240, 250, 260 and 270; a plural number of air holes 212, 222,272, 273; and a plural number of cover-attached air holes 221, 231, 241,251, 261 and 271. The channel portion 930 comprises a positioning hole710, and a container, which will be mentioned later, and channels, etc.Also, the analysis disc 902 is positioned by inserting the projection121 of the holder disc 912 into the positioning hole 710.

[0157] The structure of the channel portion 930 mentioned above will beshown in FIG. 23 attached. The embodiment of the channel portion shownin this FIG. 23 comprises a channel, so as to add a detection reagentfor analysis thereto, after separation of the serum from the wholeblood, and after extraction of nucleic acids, which are contained in thevirus within the serum.

[0158] Hereinafter, explanation will be made on an analysis operation ofthe viral nucleic acid in the case of using the whole blood as thesample. However, flows of the extraction and the analysis operation willbe shown, by referring to FIGS. 24 and 25, and the flow conditionswithin the channel portion 930 will be shown, by referring to FIGS. 26to 38.

[0159] An operator of this apparatus, first of all, injects the reagentsinto the respective reagent containers 320, 330, 340, 350, 360 and 370,from the upper cover 920 of the analysis disc 902 through the injectionopenings 220, 230, 240, 250, 260 and 270, separately, and thereafterkeeps them covered. The operator mounts the analysis discs 902 onto theholding disc 912 in a necessary number thereof, after injecting thereagents therein, depending upon that necessary number of analysis.

[0160] Next, the whole blood, being collected by means of a vacuum bloodcollection tube, etc., is injected into the sample container 310 fromthe sample injection opening 210 (see FIG. 26).

[0161] After injection of the whole blood 501, the holder disc 912 isrotated by means of the motor 911. The whole blood injected into thesample container 310 flows into an outer peripheral side thereof due tothe function of the centrifugal force, which is generated accompanyingwith the rotation of the holder disc 912, thereby filling up withininsides of a blood cell storage container 311 and a serum quantificationcontainer 312, on the other hand the whole blood in excess flows into awhole blood disposal container 315 from an overflow fine tube channel313 through an overflow thick tube channel 314 (see FIG. 27). This wholeblood disposal container 315 has an air channel 318 for use of disposalof whole blood, and further there is an air hole 212 for use of disposalof whole blood at the position corresponding to the most-interiorportion of the whole blood disposal air channel 318 on the upper cover920. For this reason, the air can freely go in and out therethrough. Theconnection portion is expanded abruptly, covering from the overflow finetube channel 313 to the overflow thick tube channel 314, and it lieswithin the most-interior side of the overflow fine tube channel 313 (atthe radius position 601), therefore the whole blood is cut out at theconnection portion under the condition where it fills up within theoverflow fine tube channel 313. Accordingly, in an inner side from theradius portion 601 in the figure, the liquid cannot lie therein, forthis reason, also a liquid surface in the serum quantification container312 comes up to this radius position 601. Further, the whole blood flowsalso into a serum capillary tube 316, which is divided from the serumquantification container 312, and also herein the most inner potion ofthe whole blood lies at the radius position 601.

[0162] Further when the holder disc continues the rotation thereof, thewhole blood 501 is separated into the blood cells and the serum (i.e.,the centrifugal separation), and thereby the blood cell 502 moves intothe blood cell storage container 311 at the outer peripheral side, whilethe serum quantification container 312 being filled up with only theserum 503 therein (see FIG. 28).

[0163] However, in the series of operations for separating the serummentioned above, the air holes 221, 231, 241, 251, 261 and 271 of therespective reagent containers are under the condition that they areclosed up with the covers thereof, so that the air cannot come intherefrom.

[0164] On the other hand, though each of the reagents tries to flow outfrom the outer-peripheral side thereof due to the centrifugal forceapplied, however it cannot flow outside, because within the reagentcontainer, into which no air can enter therein, the pressure beinglowered maintains the balance with the centrifugal force. However, inaccordance with rise-up in the centrifugal force with an increase of therotation speed, the pressure within this reagent container is loweredgradually, and when it comes to be equal or less than the saturationvapor pressure of the reagent, bubbles are generated in an insidethereof. Then, forming such a channel structure for turning the reagentflowing out from the outer peripheral side of each the reagent containerinto the inside thereof, once, as shown in FIG. 26 attached (i.e.,return channels 322, 332, 342, 352, 362 and 372), enables to suppressthe lowering of the pressure within the reagent container, and therebyto prohibit the bubbles from generating in the inside thereof. In thismanner, each the reagent will not flow, while being held within thereagent container, during the time-period of the separation operation ofserum.

[0165] When completing the separation operation of serum throughrotating thereof for a predetermined time, the analysis disc 902 isstopped, and a part of the serum 503 within the serum quantificationcontainer 312 moves into an inside of the serum capillary tube 316 dueto the surface tension (i.e., the capillary movement). Thus, it entersup to a mixing portion inlet, i.e., the connecting portion between amixing portion 410 and the serum capillary tube 316, thereby filling upthe inside of the serum capillary tube 316 therewith.

[0166] Hereinafter, the punching machine 913 opens a hole one by one, onthe cover of the air holes, being provided on an upper portion of eachof the reagent containers, and thereafter the motor 11 is rotated,thereby letting the each agent to flow due to the centrifugal force. Asis shown on the cross-section view of the analysis disc, the reagentinjection openings (i.e., 240, 250, and 260) and the air holes (i.e.,241, 251, and 261) are provided on the upper cover 920, corresponding tothe upper portion of each of the reagent containers, and those air holesare closed up with the cover. Then, the punching machine 913 opens theholes on this cover, thereby bringing it under the condition that theair can come into the inside of the said reagent container.

[0167] Hereinafter, operations will be shown, after the completion ofthe separation of serum.

[0168] Into the eluting solution container 320 is injected an elutingsolution 521 for lysing protein of the virus within the serum therein,separately. Namely, when the motor 911 is rotated after the punchingmachine 913 opens the hole on the cover of the lysis solution air hole221, the lysis solution 521 flows from the lysis solution container 320through the return channel 322 into the mixing portion 410 due to thefunction of the centrifugal force. Also, since the most-inner side ofthe serum within the serum quantification container 312 (i.e., it lieson the radius position 601 when completing the separation of serum) lieswithin an inner periphery side than an inlet 411 of the mixing portion(on the radius position 602), because of the head difference due to thecentrifugal force, the serum within the serum quantification container312 and the serum capillary tube 316 flows from the mixing portion inlet411 into the mixing portion 410 (see FIG. 29). Further, this mixingportion 410 is made up with a material, within which the serum and thelysis solution can be mixed up, such as a porous filter or fiber ofresin, glass or paper, etc., or projections of silicon or metal, etc.,which is manufactured through etching or mechanical machining, forexample.

[0169] Then, the serum and the lysis solution, being mixed within themixing portion 410 mentioned above, flows into a reactor container 420(see FIG. 30). In this reactor container 420, there is provided anairflow passage 423 for the reactor container, and further is providedan airflow passage 222 for the reactor container at the positioncorresponding to the most-inner peripheral portion of the reactorcontainer airflow passage 423 on the upper cover 920, therefore the aircan comes in and out, freely within the inside and the outside thereof.And, the branch portion 317 from the serum quantification container 312to the serum capillary tube 316 (at the radius portion 603) lies withinthe inner periphery side than the mixing portion inlet 411 (at theradius position 602) mentioned above, therefore due to the effect ofso-called the siphon, all the serum within the serum capillary tube 316flows out into the mixing portion 410. On the other hand, since theserum within the serum quantification container 312 flows into the serumcapillary tube 316 due to the centrifugal force, the serum continues toflow out into the mixing portion 410 until when the liquid surface ofthe serum reaches to the branch portion 317 (at the radius position603). And, at a time point when the liquid surface of the serum reachesto the branch portion 317, the air is mixed into the serum capillarytube 316, so as to bring the inside thereof empty, thereby the serumstops flowing thereof. Namely, all the serum lying from the radiusportion 601 to the radius position 603 at the time of completion ofseparation thereof, i.e., the serum within the serum quantificationcontainer 312, the overflow fine tube channel 313, and the serumcapillary tube channel 316, flows into the mixing portion 410, therebybeing mixed up with the lysis solution.

[0170] In the reactor container 420 react the mixed serum and the lysissolution with each other. Since the liquid surface lies within an outerperiphery side than the most-inner periphery portion (i.e., the radiusposition 604) of a flow passage 421 of reacting liquid in the reactorcontainer 420 after the mixture of the serum and the lysis solutionflows to move into the reactor container 420, the mixture cannot crossover the most-inner periphery portion of the reacting liquid flowpassage, therefore the mixture is held within the reactor container 420during the rotation thereof.

[0171] The lysis solution functions to dissolve the membrane of thevirus and/or the bacteria within the serum, thereby elute the nucleicacid therefrom, and it accelerate absorption of the nucleic acid on anucleic acid combining material 301. As such the reagent as wasmentioned may be used guanidine-hydrochloric acid, in particular forelution and absorption of the DNA, on the other hand guanidinethiocyanate for the RNA, and as such the combining material of thenucleic acid may be used a porous material, such as quartz or glass,etc., or a fiber filter or the like, for example.

[0172] Next, after the serum and the lysis solution are held within thereactor container 420, the motor 911 is stopped, holes are opened on thecover of the addition liquid air hole 231 by means of the punchingmachine 913, for supplying the air into the addition liquid container330, and then the motor 911 is rotated again. Then, due to the functionof the centrifugal force, an addition liquid 531 flows from the additionliquid container 330 through the addition liquid return passage 332 intothe reactor container 420, thereby bringing the liquid surface of themixture liquid within the reactor container 420 into the inner peripheryside thereof (see FIG. 31). Thereafter, when this liquid surface reachesup to the most-inner periphery portion of the reacting liquid passage421 (i.e., the radius position 604), the mixture liquid flows out,exceeding the most-inner periphery portion of the reacting liquid flowpassage, through a combined flow passage 422, into the nucleic acidcombining material 310. As such the addition liquid as was mentioned maybe used the lysis solution mentioned above, for example.

[0173] Further, depending upon the sample, it sometimes happens that themixture liquid flows to move within the reacting liquid flow passage 421due to the phenomenon of capillary tube, even if the mixture liquid issuperior in wetability on a wall surface and/or under the condition ofstopping thereof, and in such the instance, the addition liquid is notneeded.

[0174] When the lysis solution and the serum pass or penetrate throughthe nucleic acid combining material 301 in this manner, the nucleic acidis absorbed onto the nucleic acid combining material 301, while theliquid (i.e., the mixture liquid) flows into a detector container 450.This mixture liquid is sufficiently larger than the volume of thedetector container 450, into which it flows into, in a liquid amountthereof, and therefore it flows out, exceeding the most-inner peripheryportion of a waste liquid flow passage 452 (i.e., the radius position607) into a waste liquid container 460 (see FIG. 32).

[0175] On the other hand, the mixture liquid within the reactorcontainer 420 flows out gradually, from the combined flow passage 422through reacting liquid flow passage 421. However, since the branchportion (i.e., at the radius position 605) from the reactor container420 to the reacting liquid flow passage 421 is located within an innerperiphery side than the combined flow passage 422 (i.e., at the radiusposition 606), all the mixture liquid within the reactor container 420flows into the combined flow passage 422 due to the siphon phenomenon(see FIG. 33). The liquid flowing into the detector container 450,passing through the nucleic acid combining material 310, is also in thesame manner as was mentioned, i.e., all the liquid within the detectorcontainer 450 flows out into the waste liquid container 460 through thewaste liquid flow passage 452, due to the siphon phenomenon, too, sincethe branch portion from the detector container 450 to the waste liquidflow passage 452 (i.e., at the radius position 608) is located withinthe inner periphery side than an exit to the waste liquid flow passage452 into the waste liquid container 460.

[0176] Next, the motor 911 is stopped, and holes are opened by means ofthe punching machine 913, on the cover of the first washing liquid airhole 241, for supplying the air into the first washing liquid container340. Thereafter, when the motor 911 is rotated again, due to thefunction of the centrifugal force, the first washing liquid 541 flowsfrom the first washing liquid container 340 through the first washingliquid return flow passage 342 into the nucleic acid combining material301, thereby flowing out unnecessary components, such as the protein,etc., attaching onto the nucleic acid combining material 301, forexample (see FIG. 34). Further, as the first washing liquid may be used,for example, the lysis solution mentioned above, or a liquid beinglowered in the concentration of salt of the said lysis solution. Also, aliquid amount of the first washing liquid 541 is sufficient larger thanthe volume of the detector container 450, and therefore it flows outinto the waste liquid container 460, exceeding the most-inner peripheryportion of the water liquid flow passage 452 (i.e., at the radiusposition 607), (see FIG. 34).

[0177] Furthermore, all the liquid within the detector container 450flows out into the waste liquid container 460 through the waste liquidflow passage 452, due to the siphon phenomenon too, (see FIG. 35).

[0178] Hereinafter, the same washing operations are repeated in a pluralnumber thereof. For example, following those for the first washingliquid, a hole is opened on the cover of the second rinsing liquid airhole 241 by means of the punching machine 913 for supplying the air intothe second washing liquid container 350, and thereafter the motor 911 isrotated, again. With this, the unnecessary components, such as salt,etc, attaching onto the nucleic acid boding material 301, is flowed out(see FIG. 36). Further, such the washing liquid as was mentioned may beused ethanol, or a water solution of ethanol, for example. Also, thiswashing may be repeated further, in the same manner, depending upon thenecessity thereof.

[0179] As was mentioned in the above, after following of washing out thenucleic acid combining material 301, thereby bringing it under thecondition of absorbing only the nucleic acid in the nucleic acidcombining material 301, the process is shifted into an eluting processof the nucleic acid.

[0180] Namely, in this eluting process of the nucleic acid, under thecondition that the motor is stopped, a hole is opened by means of thepunching machine 913, on the cover of the eluting solution air hole 261for supplying the air into the eluting solution container 360, andfurther a hole is opened on the cover of the air hole for use of wastingthe eluting solution, for communicating an eluting solution wastecontainer 470 with an outside thereof. And, the motor 911 is rotated,again, thereby flowing the eluting solution into the nucleic acidcombining material 301 (see FIG. 36). This eluting solution is a liquidused for eluting the nucleic acid from the nucleic acid combiningmaterial 301, and it may a water or a water solution, which is adjustedfrom 7 to 9 in pH, for example. In particular, for the purpose ofbringing the nucleic acid to elute easily therein, it is preferable tobe heated it up to be equal or higher than 40° C. For this heating, alight may be irradiated upon from the above of the eluting solutioncontainer 360, with using the upper optical apparatus 914 shown in FIG.21 mentioned above.

[0181] After passing or penetrating through the nucleic acid combiningmaterial 301, the eluting solution 561 flows into the detector container450 (see FIG. 37). A liquid amount of this eluting solution is smallerthan the volume of the detector container 450, and therefore the liquidwithin the detector container 450 (i.e., at the radius position 610 ofthe liquid surface) cannot cross over the most-inner periphery portionof the waste liquid flow passage 452 (i.e., at the radius position 607),thereby being held within the detector container 450.

[0182] Next, under the condition that the motor is stopped, a hole isopened by means of the punching machine 913, on the cover of thedetection liquid air hole 271 for supplying the air into the detectionliquid reservoir container 370, and thereafter the motor 911 is rotated,again, thereby letting the detection liquid 571 to flow into thedetector container 450 (see FIG. 38). This detection liquid may be areagent for amplifying the nucleic acid to be detected, such as deoxynucleoside triphosphate or DNA synthetic enzyme, for example, and it mayinclude fluorescence reagent therein. Further, depending upon theamplifying method, it may be possible to heat it up, with using theupper portion optical apparatus 914 shown in FIG. 21 mentioned above,thereby irradiating the light thereupon from the above of the detectorcontainer 450.

[0183] Continuously, also moving the lower portion optical apparatus 915also shown in FIG. 21 mentioned above below the detector container 450,thereby an amount of emission of fluorescence is detected, for example.

[0184] By the way, at the time of punching, the heating, and thedetecting, it is necessary to stop the holder disc 912 at thepredetermined position. Then, according to the present embodiment, asshown in FIG. 39, a projection 917 is provided on the holder disc 912for positioning thereof, and it is detected by means of a positiondetector 916, thereby detecting the rotation position of the holderdisc. Also, the controller 918 controls the rotation of the motor 911,the rotation and the vertical movement of the punching machine 913, andthe rotation, the irradiation and the detection of the upper portionoptical apparatus 914 and the lower portion optical apparatus 915.

[0185]FIG. 40 shows the operation timing of the punching machine 913,for example. Namely, after completion of flowing of the whole blood andthe respective reagents, the holder disc 912 is lowered in the rotationspeed, and is maintained at a low rotation for positioning. When theposition detector 916 detects the positioning projection 917, the holderdisc 912 is stopped, and then the punching machine 913, after comingdown to open the holes on the covers of the air holes for the respectivereagent reservoir containers, goes up again. After this punching, theholder disc 912 is rotated at a low speed, so that no reagent flows outfrom the reagent reservoir containers after the punching, and it isstopped at the position of the next analysis disc, i.e., after rotatingby sixty (60) degree when the analysis discs are mounted in six (6)pieces, and it repeats the same punching operation as was mentionedabove. Further, the mounting positions of the analysis discs may besearched through a reflection light, which can be obtained byirradiation of the light from an optical window of the holder disc ontothe channel portions 930 by means of the lower portion optical apparatus915, for example. And, after completion of punching of all the analysisdiscs, the holder disc is rotated at a high speed, thereby bringing thereagents to flow to move.

[0186] As was mentioned in the above, according to the presentinvention, there is no necessity of provision of such the valves on theway of the flow passages, for controlling the flow of the respectivereagents, as in the conventional art, nor no liquid remains within thevalve portion on the way of the flow passages, therefore it is possibleprevent from the contamination by the reagent in the pre-processing,thereby enabling extraction of a specific component, such as the nucleicacid or the like within the liquid sample, for example, at a highpurity, so as to analyze the specific component, such as the nucleicacid or the like at high accuracy.

[0187] Further, as was mentioned previously, it is preferable to applythe present invention, in particular, into the analysis apparatus forrefining the nucleic acid and/or for analyzing the extracted nucleicacid, i.e., the genes, and/or the analysis method therefore, however itmay be applied into the apparatus and the method for refining chemicalmaterials other than that. Namely, in more details, according to thepresent invention, it is possible to construct the analysis apparatusand the method thereof for analyzing the chemical materials, such asproteins and/or amino acids, etc., for example.

INDUSTRIAL APPLICABILITY

[0188] The present invention can be applied into the apparatus forrefining the nucleic acid from a sample containing the nucleic acidtherein. In particular, it can be also applied into, such as anautomatic refining apparatus and the refining method thereof, beingsuitable for separating the nucleic acid from the materials coexistingwith the nucleic acid, by utilizing the centrifugal force, and furthercan be applied into the refining apparatus of the chemical materials,etc. Also, it can be applied into a gene analysis apparatus comprisingsuch the apparatus therein.

1. A nucleic acid refining apparatus for refining nucleic acid from asample containing the nucleic acid therein, comprising: means forseparating a liquid containing the nucleic acid therein from said samplethrough centrifugal force; means for transferring a reagent through thecentrifugal force; means for producing a mixture liquid of said reagenttransferred through the centrifugal force and a solution containing saidnucleic acid therein; a carrier for capturing said nucleic acid; meansfor transferring said mixture liquid to said carrier through thecentrifugal force; heating means for heating said carrier; and a holdingmeans for separating and holding the reagent containing said nucleicacid eluting from said carrier, separating from other reagent, throughdifferent centrifugal.
 2. A nucleic acid refining apparatus for refiningnucleic acid from a sample containing the nucleic acid therein,comprising: means for separating a liquid containing the nucleic acidtherein from said sample through centrifugal force; reagent holdingmeans for holding a reagent therein; means for transferring said reagentfrom said reagent holding means through the centrifugal force; means forproducing a mixture liquid of said reagent transferred through thecentrifugal force and a solution containing said nucleic acid therein; acarrier for capturing said nucleic acid; means for transferring saidmixture liquid to said carrier through the centrifugal force; heatingmeans for heating said carrier; and a holding means for separating andholding the reagent containing said nucleic acid eluting from saidcarrier, separating from other reagent, through different centrifugal.3. A nucleic acid refining apparatus for refining nucleic acid from asample containing the nucleic acid therein, comprising: a device having:means for separating a liquid containing the nucleic acid therein fromsaid sample through centrifugal force; means for transferring a reagentthrough the centrifugal force; means for producing a mixture liquid ofsaid reagent transferred through the centrifugal force and a solutioncontaining said nucleic acid therein; a carrier for capturing saidnucleic acid; means for transferring said mixture liquid to said carrierthrough the centrifugal force; heating means for heating said carrier;and a holding means for separating and holding the reagent containingsaid nucleic acid eluting from said carrier, separating from otherreagent, through different centrifugal; and a supply means for supplyingsaid reagent from an outside of said device.
 4. A nucleic acid refiningapparatus for refining nucleic acid from a sample containing the nucleicacid therein, comprising: a round front surface cover having a hole,which is sealed by a rubber at one end thereof; a gap defined betweensaid front surface cover; a first round disc lying in said gap andhaving a separation gel for separating a solution including nucleic acidtherein from said sample and a groove for quantification of saidsolution; a second round disc having: a reagent container containing areagent therein; a flow passage; a carrier for combining the nucleicacid thereon; an elution container for accumulating an eluting solution,being obtained from said reagent after eluting the nucleic acid therein;and a waste liquid container, being provided following said elutingsolution container, for accumulating the reagent other than said elutingsolution, and having a flow passage being opened into an outside; and around reverse surface cover having a heating body, wherein a device isbuilt up with said reverse surface cover, said second disc, said firstdisc and said front surface cover, being piled up sequentially, and hasa punching portion, through which said solution and said reagent canmove in a direction of thickness of said device, by punching a hole at apredetermined position within said device.
 5. A nucleic acid refiningapparatus as described in the claim 4, wherein a “U” shaped flow passageis provided between said solution container of said second disc and saidwaste liquid container.
 6. A nucleic acid refining apparatus asdescribed in the claim 4, wherein a branch passage, being divided intothe direction of thickness of said device, is provided between saidsolution container of said second disc and said waste liquid container.7. A nucleic acid refining apparatus as described in the claim 4,wherein a filter, having penetrability lower than that of said carrier,is provided between said solution container of said second disc and saidwaste liquid container.
 8. A method for refining nucleic acid from asample containing the nucleic acid therein, comprising the followingsteps of: a step for separating a solution containing nucleic acidtherein from said sample through centrifugal force, with using aseparation gel, within a first gap provided within an inside of adevice, being formed with laminating a plural number of round discs; astep for quantification of said solution with provision of a first holewithin said device; a step for transferring said quantified solutioninto a flow passage, as a second hole formed within said device, withprovision of a second hole within said second device; a step fortransferring a combining liquid of a first reagent into said flowpassage through centrifugal force, with provision of a third hole withinsaid device; a step for producing a mixture liquid of said solutionquantified within said flow passage and said combining liquid; a stepfor passing said combining liquid through said carrier for capturingsaid nucleic acid thereon through centrifugal force, therebytransferring it into a waste liquid container as a third gap formedwithin said device; a step for transferring a rinsing liquid of a secondreagent into said flow passage through the centrifugal force, withprovision of a fourth hole within said device; a step for making saidrinsing liquid pass through said carrier through the centrifugal force,thereby transferring it into said waste liquid container; a step fortransferring an eluting solution of a third reagent into said flowpassage through the centrifugal force, with provision of a fifth holeformed within said device; a step for letting said carrier to hold saideluting solution therein, and heating said carrier; a step fortransferring said eluting solution containing said nucleic acidseparated from said carrier therein into an eluting solution containerformed within a flow passage following said carrier, through thecentrifugal force, thereby holding the eluting solution thereinseparating from other reagents; and a step for collecting said elutingsolution from said eluting solution container, from an outside of saiddevice.
 9. A nucleic acid refining structure, being formed to berotatable, comprising: a supply portion, through which is supplied aliquid including nucleic acid therein; a nucleic acid capture portion,onto which is captured the nucleic acid within said supplied liquid; awashing liquid supply portion, through which a washing liquid issupplied to said nucleic acid capture portion; a waste portion, intowhich is wasted said washing liquid flowing through said nucleic acidcapture portion; an eluting solution supply portion, through which aneluting solution is supplied into said nucleic acid capture portion; andan eluting solution holder portion for holding the eluting solutionincluding the nucleic acid therein, being captured on said nucleic acidcapture portion once and separated therefrom, after flowing through saidnucleic acid capture portion, wherein said eluting solution hold portionis formed within a flow passage communicating between said nucleic acidcapture portion and said waste portion.
 10. A nucleic acid refiningstructure, being formed to be rotatable, comprising: a supply portion,through which is supplied a liquid including nucleic acid therein; anucleic acid capture portion, onto which is captured the nucleic acidwithin said supplied liquid; a washing liquid supply portion, throughwhich a washing liquid is supplied to said nucleic acid capture portion;a waste portion, into which is wasted said washing liquid flowingthrough said nucleic acid capture portion; an eluting solution supplyportion, through which an eluting solution is supplied into said nucleicacid capture portion; and an eluting solution holder portion for holdingthe eluting solution including the nucleic acid therein, being capturedon said nucleic acid capture portion once and separated therefrom, afterflowing through said nucleic acid capture portion, wherein said elutingsolution holder portion is formed in a downstream of said nucleic acidcapture portion, while said waste portion in a downstream of saideluting solution holder portion.
 11. A nucleic acid refining structure,being formed to be rotatable, comprising: a supply portion, throughwhich is supplied a liquid including nucleic acid therein; a nucleicacid capture portion, onto which is captured the nucleic acid withinsaid supplied liquid; a washing liquid supply portion, through which awashing liquid is supplied to said nucleic acid capture portion; a wasteportion, into which is wasted said washing liquid flowing through saidnucleic acid capture portion; an eluting solution supply portion,through which an eluting solution is supplied into said nucleic acidcapture portion; and an eluting solution holder portion for holding theeluting solution including the nucleic acid therein, being captured onsaid nucleic acid capture portion once and separated therefrom, afterflowing through said nucleic acid capture portion, wherein said elutingsolution holder portion is formed on an outer periphery side of saidnucleic acid capture portion, while said waste portion on an outerperiphery side of said nucleic acid holder portion.
 12. A nucleic acidrefining structure, being formed to be rotatable, comprising: a supplyportion, through which is supplied a liquid including nucleic acidtherein; a nucleic acid capture portion, onto which is captured thenucleic acid within said supplied liquid; a washing liquid supplyportion, through which a washing liquid is supplied to said nucleic acidcapture portion; a waste portion, into which is wasted said washingliquid flowing through said nucleic acid capture portion; an elutingsolution supply portion, through which an eluting solution is suppliedinto said nucleic acid capture portion; an eluting solution holderportion for holding the eluting solution including the nucleic acidtherein, being captured on said nucleic acid capture portion once andseparated therefrom, after flowing through said nucleic acid captureportion; and a waste liquid flow passage communicating between saideluting solution holder portion and said waste portion, wherein saidwaste liquid flow passage comprises a holder portion communicationportion being communicated with an area, being located on an outerperiphery side rather than an area on a most-inner periphery side ofsaid eluting solution holder portion, an inner periphery side areaportion, being located in a downstream of said communication portion andin an inner periphery side rather than said communication portion, and awaste portion communication portion, being located in a downstream ofsaid inner periphery side area portion and being communicating with saidwaste portion, being located in an outer periphery side rather than saidinner periphery side area portion.
 13. A nucleic acid refiningstructure, being formed to be rotatable, comprising: a supply portion,through which is supplied a liquid including nucleic acid therein; anucleic acid capture portion, onto which is captured the nucleic acidwithin said supplied liquid; a washing liquid supply portion, throughwhich a washing liquid is supplied to said nucleic acid capture portion;a waste portion, into which is wasted said washing liquid flowingthrough said nucleic acid capture portion; an eluting solution supplyportion, through which an eluting solution is supplied into said nucleicacid capture portion; an eluting solution holder portion for holding theeluting solution including the nucleic acid therein, being captured onsaid nucleic acid capture portion once and separated therefrom, afterflowing through said nucleic acid capture portion; and a waste liquidflow passage communicating between said eluting solution holder portionand said waste portion, wherein a connection portion between saideluting solution holder portion and said waste liquid flow passage isformed in an outer periphery side rather than a most-inner peripheryportion of said waste liquid flow passage, and in an inner peripheryside rather than a most-outer periphery portion of said waste liquidflow passage.
 14. A nucleic acid refining structure, being formed to berotatable, comprising: a supply portion, through which is supplied aliquid including nucleic acid therein; a nucleic acid capture portion,onto which is captured the nucleic acid within said supplied liquid; afirst reagent supply portion, through which a first reagent is suppliedto said nucleic acid capture portion; a waste portion, into which iswasted said first reagent flowing through said nucleic acid captureportion; a second reagent supply portion, through which a second reagentis supplied to said nucleic acid capture portion, having a function ofreleasing said nucleic acid captured on said nucleic acid captureportion therefrom, being larger than that of said first reagent; asecond reagent holder portion for separating the nucleic acid capturedon said nucleic acid capture portion therefrom, thereby to hold saidsecond reagent contained within an inside thereof; and a waste liquidflow passage communicating between said second reagent holder portionand said waste portion, wherein an area located on said waste flowpassage between a most-inner periphery portion and a connection portionwith said second reagent holder portion, and an area located on an outerperiphery side than said most-inner periphery portion of said secondreagent holder portion are formed, so that a total volume of them issmaller than that of said first reagent to be supplied, while beinglarger than that of said second reagent to be supplied.
 15. A nucleicacid refining structure, being formed to be rotatable, comprising: asupply portion, through which is supplied a liquid including nucleicacid therein; a nucleic acid capture portion, onto which is captured thenucleic acid within said supplied liquid; a washing liquid supplyportion, through which a washing liquid is supplied to said nucleic acidcapture portion; a waste portion, into which is wasted said washingliquid flowing through said nucleic acid capture portion; an elutingsolution supply portion, through which an eluting solution is suppliedinto said nucleic acid capture portion; an eluting solution holderportion for holding the eluting solution including the nucleic acidtherein, being captured on said nucleic acid capture portion once andseparated therefrom, after flowing through said nucleic acid captureportion; and a waste liquid flow passage communicating between saideluting solution holder portion and said waste portion, wherein an arealocated on said waste flow passage between a most-inner peripheryportion and a connection portion with said eluting solution holderportion and an area located on an outer periphery side than saidmost-inner periphery portion of said eluting solution holder portion areformed, so that a total volume of them is smaller than that of saidwashing liquid to be supplied, while being larger than that of saideluting solution to be supplied.
 16. A nucleic acid refining apparatuscomprising: a receiver portion for receiving said nucleic acid refiningstructure described in the claim 9; and a rotary driving mechanism forrotating said nucleic acid refining structure.
 17. A nucleic acidrefining apparatus as described in the claim 16, further comprising ananalysis mechanism for analyzing genes of said nucleic acid, with usingsaid nucleic acid introduced into said eluting solution holder portionof said nucleic acid refining structure.
 18. A nucleic acid refiningapparatus as described in the claim 16, further comprising a heatingdevice for heating said nucleic acid introduced into said elutingsolution holder portion of said nucleic acid refining structure.
 19. Anucleic acid refining apparatus as described in the claim 16, whereinsaid washing liquid is controlled, so as to be held within said wasteportion, being supplied from said nucleic acid capture portion throughsaid eluting solution holder portion, in an amount larger than that atotal volume of an area located on said waste flow passage between amost-inner periphery portion and a connection portion with said elutingsolution holder portion and an area located on an outer periphery sidethan said most-inner periphery portion of said eluting solution holderportion, while said eluting solution is controlled, so as to be heldwithin said eluting solution holder portion, being supplied through saidnucleic acid capture portion, in an amount smaller than that the totalvolume of the area located on said waste flow passage between themost-inner periphery portion and a connection portion with said elutingsolution holder portion and the area located on the outer periphery sidethan said most-inner periphery portion of said eluting solution holderportion.
 20. A chemical material refining structure comprising: a supplyportion, through which is supplied a liquid including a first chemicalmaterial therein; a first chemical material capture portion, onto whichis captured said chemical material within said supplied liquid; a firstreagent supply portion, through which a first reagent is supplied tosaid first chemical material capture portion; a waste portion, intowhich is wasted said first reagent flowing through said first chemicalmaterial capture portion; a second reagent supply portion, through whicha second reagent is supplied to said first chemical material captureportion, having a function of releasing said chemical material from saidfirst chemical material capture portion, being larger than that of saidfirst reagent; and a second reagent holder portion for holding saidsecond reagent including said first chemical material therein, beingcaptured on said first chemical material capture portion once and thenseparated therefrom after flowing through said first chemical materialcapture portion, wherein said second reagent holder portion is formedwithin a flow passage communicating between said first chemical martialholder portion and said waste portion.